This project aims to determine physiologically critical functions of NF-kappaB proteins and their regulators in the context of specific biologic reactions. Regulators include the inhibitory IkappaB proteins, as well as proximal activators. Research is based on the discovery of specific defects (especially of the immune system) in mice rendered deficient for various NF- kappaB proteins or their regulators. The ultimate goal is to identify critical molecular targets of the NF-kappaB factors in specific immune responses and to identify the essential signals that activate the factors. Previously we have generated mice deficient in the NF-kappaB2-locus encoded proteins p52 and its precursor p100. In addition we have generated mice deficient in both NF-kappaB1 and NF-kappaB2. Double knockout mice are completely blocked in the development of mature osteoclasts and of mature B cells. We determined in adoptive transfer experiments that the blocks in generation of mature B cells and mature osteoclasts are intrinsic to these cell lineages. We now demonstrated that NF-kappaB activation in developing osteoclasts occurs in response to RANK receptor stimulation, allowing for continued differentiation of osteoclast progenitors into mature osteoclasts. We also demonstrated that B cell development in double knockout mice is blocked during an early transitional stage of differentiation of B cells in spleen, shortly after entry from the bone marrow, resulting in a complete absence of recirculating mature B cells. The double-deficient mutant B cells are intrinsically more apoptotic than their wild-type counterparts, indicating that NF-kappaB1,2 are essential to the survival of transitional splenic B cells. This conclusions is now confirmed with mice in which the anti-apoptotic regulator Bcl-2 is expressed from a transgene in B cells; in such transgenic mice B cell development is largely, though not completely rescued. The specific contribution of NF-kappaB2 to survival could be shown to occur in response to the TNF-family member BAFF. BAFF induced the processing of the p100 precursor form of NF-kappaB2 to p52 and the processing was necessary for the extended survival of transitional B cells. These results indicate that NF-kappaB2 processing, an alternative pathway of activating NF-kappaB, is involved in survival of transitional B cells in spleen, at a stage of development when only a few of these cells are selected into the mature pool of B cells and when self-tolerance is enforced. We have also discovered that NF-kappaB1,2 contribute to the development of B cells in the bone marrow, since preB cells are partially blocked in their development in the knockout mice. This defect becomes particularly noticeable in the presence of wild-type B cells, indicating that only under such competitive conditions is the true contribution of NF-kappaB to B cell development revealed. Finally we have demonstrated that mammary gland development depends on NF-kappaB activation in mammary epithelial cells and that this involves a kinase, NIK, which is activated by the Rank receptor and which induces processing of NF-kappaB2 p100. The contribution of processing to mammary gland development is under investigation.